Chemiluminescent assays for the detection of the presence or concentration of a biological substance have received increasing attention in recent years as a fast, sensitive and easily read method of conducting bioassays. In such assays, a chemiluminescent compound is used as a reporter molecule, the reporter molecule chemiluminescing in response to the presence or the absence of the suspected biopolymer.
A wide variety of chemiluminescent compounds have been identified for use as reporter molecules. One class of compounds receiving particular attention is the 1,2-dioxetanes. 1,2-dioxetanes can be stabilized by the addition of a stabilizing group to at least one of the carbon atoms of the dioxetane ring. An exemplary stabilizing group is spiro-bound adamantane. Such dioxetanes can be further substituted at the other carbon position with an aryl moiety, preferably phenyl or naphthyl, the aryl moiety being substituted by an oxygen which is, in turn, bound to an enzyme-labile group. When contacted by an enzyme capable of cleaving the labile group, the oxyanion of the dioxetane is formed, leading to decomposition of the dioxetane and spontaneous chemiluminescence. A wide variety of such dioxetanes are disclosed in U.S. Pat. No. 5,112,960. That patent focuses on dioxetanes which bear a substituent on the adamantyl-stabilizing group, such as halo substituents, alkyl groups, alkoxy groups and the like. Such dioxetanes represent an advance over earlier-recognized dioxetanes, such as 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo]-3.3.1.1.sup.3,7 ]decan]-4-yl) phenyl phosphate, and in particular, the disodium salt thereof, generally identified as AMPPD.RTM.. The chlorine-substituted counterpart, which converts the stabilizing adamantyl group from a passive group which allows the decomposition reaction to go forward, to an active group which gives rise to enhanced chemiluminescence signal due to faster decomposition of the dioxetane anion, greater signal-to-noise values and better sensitivity, is referred to as CSPD.RTM.. Other dioxetanes, such as the phenyloxy-.beta.-D-galactopyranoside (AMPGD) are also well-known, and can be used as reporter molecules. These dioxetanes, and their preparation, do not constitute an aspect of the invention herein, per se.
Assays employing these dioxetanes can include conventional assays, such as Southern, Northern and Western blot assays, DNA sequencing, ELISA, as well as other liquid phase and mixed phase assays performed on membranes and beads. In general, procedures are performed according to standard, well-known protocols except for the detection step. In DNA assays, the target biological substance is bound by a DNA probe with an enzyme covalently or indirectly linked thereto, the probe being admixed with the sample immobilized on a membrane, to permit hybridization. Thereafter, excess enzyme complex is removed, and dioxetane added to the hybridized sample. If hybridization has occurred, the dioxetane will be activated by the bound enzyme, leading to decomposition of the dioxetane, and chemiluminescence. In solution-phase assays, the enzyme is frequently conjugated to a nucleic acid probe or immune complexed with an antibody responsive to the target biological substance, unbound components being removed, and the dioxetane added, chemiluminescence being produced by the decomposition of the dioxetane activated by the amount of enzyme present. In cases where the enzyme itself is the target, the dioxetane need only be added to the sample. Again, a wide variety of assay modalities has been developed, as disclosed in U.S. Pat. No. 5,112,960, as well as U.S. Pat. No. 4,978,614.
It has been well-known that light-quenching reactions will occur if the dioxetane decomposition occurs in a protic solvent, such as water. As the samples suspected of containing or lacking the analyte in question are generally biological samples, these assays generally take place in an aqueous environment. The light-quenching reactions therefor may substantially reduce the chemiluminescence actually observed from the decomposition of the dioxetane. In assays involving low-level detections of particular analytes, such as nucleic acids, viral antibodies and other proteins, particularly those prepared in solution or in solution-solid phase systems, the reduced chemiluminescence observed, coupled with unavoidable background signals, may reduce the sensitivity of the assay such that extremely low levels of biological substances cannot be detected. One method of addressing this problem is the addition of water-soluble macromolecules, which may include both natural and synthetic molecules, as is disclosed in detail in U.S. Pat. No. 5,145,772. The disclosure of this patent is incorporated herein, by reference. To similar effect, U.S. Pat. No. 4,978,614 addresses the addition of various water-soluble "enhancement" agents to the sample, although the patent speaks to the problem of suppressing non-specific binding reactions in solid state assays. In U.S. Pat. No. 5,112,960, preferred water-soluble polymeric quaternary ammonium salts such as poly(vinylbenzyltrimethylammonium chloride) (TMQ) poly(vinylbenzyltributylammonium chloride) (TBQ) and poly(vinylbenzyldimethylbenzylammonium chloride) (BDMQ) are identified as water-soluble polymeric quaternary ammonium salts which enhance chemiluminescence and provide greater sensitivity by increasing the signal-to-noise ratio. Similar phosphonium and sulfonium polymeric salts are also disclosed.
This enhancement is achieved, at least in part, through the formation of hydrophobic regions in which the dioxetane oxyanion is sequestered. Decomposition in these hydrophobic regions enhances chemiluminescence, because water-based light quenching reactions are suppressed. Among the recognized water-soluble quaternary polymer salts employed, TBQ provides unexpectedly superior enhancement, through this hydrophobic region-forming mechanism.
The chemiluminescent enhancement achieved by the addition of water-soluble polymeric substances such as ammonium, phosphonium and sulfonium polymeric salts can be further improved by the inclusion, in the aqueous sample, of an additive, which improves the ability of the quaternary polymeric salt to sequester the dioxetane oxyanion and the resulting excited state emitter reporting molecule in a hydrophobic region. Thus, the combination of the polymeric quaternary salt and the additive, together, produce an increase in enhancement far beyond that produced separately by the addition of the polymeric quaternary salt, or the additive, which, when a surfactant or water-soluble polymer itself, may enhance chemiluminescence to a limited degree. The synergistic combination of the polymeric quaternary salt and additives gives enhancement effects making low-level, reliable detection possible even in aqueous samples through the use of 1,2-dioxetanes. The polymeric quaternary salts, coupled with the additives, are sufficiently powerful enhancers to show dramatic 4 and 5-fold increases at levels below 0.005 percent down to 0.001 percent. Increased signal, and improved signal/noise ratios are achieved by the addition of further amounts of the polymeric quaternary salt, the additive, or both, in amounts up to as large as 50 percent or more. In general, levels for both polymeric quaternary salt and additive can be preferably within the range of 0.01-25 percent, more preferably from 0.025-15 percent by weight. The details of this improvement are disclosed in U.S. application Ser. No. 08/031,471 which issued as U.S. Pat. No. 5,547,836 on Aug. 20, 1996 which is incorporated herein by reference.
U.S. Pat. No. 5,208,148 describes a class of fluorescent substrates for detection of cells producing the glycosidase enzyme. The substrate is a fluorescein diglycoside which is a non-fluorescent substrate until hydrolyzed by glycosidase enzyme inside a cell to yield a fluorescent detection product excitable between about 460 nm and 550 nm. The fluorescent enzymatic hydrolysis products are specifically formed and adequately retained inside living cells, and are non-toxic to the cells. The substrates can penetrate the cell membrane under physiological conditions. Therefore, the invention permits analysis, sorting and cloning of the cells and monitoring of cell development in-vitro and in-vivo. However, these fluorescent products are detected in the single cells and within specific organelles of single cells only after the spectral properties of the substrates are excited by an argon laser at its principle wavelengths.
Known fluorescent emitters have been used with dioxetanes in bioassays. U.S. Pat. Nos. 4,959,182 and 5,004,565 describe methods and compositions for energy transfer enhancement of chemiluminescence from 1,2-dioxetanes. These patents utilize a fluorescent micelle comprising a surfactant and a fluorescent co-surfactant which exists in the bulk phase of the buffer solution used. The fluorescent co-surfactant is present in a form capable of energy transfer-based fluorescence at all times. In contact with a solid phase containing an enzyme-labeled ligand binding pair, the fluorescent moiety tends to remain associated with the micelle in the bulk phase. If any fluorescent co-surfactant is deposited on the solid phase, this occurs indiscriminately, in areas containing the immobilized ligand binding pair, and in areas which do not contain said pair. Thus a problem results in that the fluorescent emitters never are, or do not remain associated with the immobilized enzyme conjugate. Thus the close proximity needed for energy transfer from the dioxetane to the fluorescent emitter is not efficient. Further because the fluorescent emitters can be deposited anywhere on the solid phase matrix, this method does not allow for specificity when used in bound assay. The majority of the examples in the '182 and '565 patents are solution phase enzyme assays or chemical triggering experiments not utilizing enzymes. These examples are better matched to the bulk phase co-micelle as a means to promote the proximity of the dioxetane anion product with the energy accepting fluorescent surfactant. The only example of a solid phase assay occurs at columns 29 and 30. This ELISA assay shows that light is produced on a well surface over the range of 112 ng to 1.3 ng of S-antigen. However, there are no control experiments showing light production from the same dose-response experiment, but using dioxetane and CTAB surfactant in the absence of fluorescent co-surfactant. Thus one cannot determine how efficient the energy transfer at the solid surface actually is. Certainly, however, this fluorescent co-surfactant is not a non-fluorescent enzyme substrate such as AttoPhos.TM.. Thus the present invention, wherein a fluorescent energy acceptor is produced directly, and locally on a surface, by the same enzyme which catalytically decomposes the dioxetane energy donor, is not suggested by these art references.
There are several basic problems which relate to fluorescent substrates used in surface or blotting experiments. One is that the excitation of the dephosphorylated chromophore has to be performed with a laser or a lamp with a filter or a monochromator. These light sources are not only cumbersome, but increase the expense of the assay. This necessary and key excitation step which is accomplished with UV/blue light results in a second problem which is autofluorescence of the membrane or surface and other solid supports which ordinarily contain fluorescent brighteners and other excitable fluorophores, as well as exciting chromophores contained in the biological sample (i.e., proteins and nucleic acids). Such fluorescent signal of the surface or membrane support and sources other than the dephosphorylated or activated substrate, contribute to unacceptable levels of background which substantially lower the sensitivity and specificity of the assay so that substrates such as these cannot be used.
Known fluorescent emitters have been used with dioxetanes in nonbound assays. However, a problem results in that the fluorescent emitters don't stay associated with the enzyme conjugate. Therefore, the close proximity needed for the energy transfer from the dioxetane to the fluorescent emitter is not possible. Further, because the fluorescent emitters don't stay associated with the enzyme conjugate, the emitters do not allow for specificity when used in bound assays.
Therefore, notwithstanding the advances in chemiluminescence technology addressed by the above assays, it remains a goal of the industry to provide chemiluminescent assays providing overall more intense signals, thus having greater sensitivity and specificity without the use of expensive, cumbersome lasers or lamps, to determine the presence, concentration or both of a biological substance in a sample. 1,2-dioxetane compounds have already been developed which show excellent potential as reporter molecules for such chemiluminescent assays. However, it is still necessary to improve upon the sensitivity and specificity of the chemiluminescence of the 1,2-dioxetane molecules by providing an efficient fluorescent acceptor emitter which stays in close contact with the dioxetane to thereby allow for the necessary energy transfer, and further, to allow for sensitive and specific determination of the target.